Stem cells can now give rise to virtually all cell types present in myocardium, offering the prospect of rebuilding the injured heart from its component parts. Our group has recently created human myocardium in the hearts of rats and mice, by transplanting cardiomyocytes derived from human embryonic stem cells (huESCs). Unlike mouse or rat cardiomyocytes, the human cells are highly proliferative, raising the possibility for exponential growth after implantation. Furthermore, newly developed techniques for directed differentiation of cardiomyocytes from hESCs (up to 50% of hESC progeny), coupled with the ability to genetically select human cardiomyocytes at ~98% purity, offer large numbers of highly purified cells for basic and preclinical research. Although promising, several obstacles must be overcome to translate these findings to a therapeutic benefit. In particular, the survival of the cells in the infarcted heart must be improved, their proliferation after implantation needs to be stimulated in a controllable fashion, and we need to prevent scar tissue from blocking their electromechanical integration with host myocardium. Experiments in Aim 1 test the hypothesis that interventions known to block ischemic or apoptotic cell death will reduce death of huESC-derived cardiomyocytes after transplantation. High throughput models will be developed to screen multiple candidates, and the most promising interventions will be tested for their ability to increase formation of human myocardium and restore contractile function in the infarcted rat heart. Aim 2 builds on our recent finding that huESC-derived cardiomyocyte proliferation requires PI-3 kinase/Akt and can be stimulated by IGF-1. We will develop systems to control human cardiomyocyte proliferation after transplantation using small molecules that activate IGF-1 receptor or FGF receptor signaling pathways in transgenic human cardiomyocytes. We then will determine whether chemically induced signaling through growth factor receptors will increase formation of human myocardium and improve ventricular function post- infarction. Aim 3 tests the hypothesis that the matricellular protein thrombospondin-2 (Tsp2), known to promote fibrosis and antagonize angiogenesis around biomaterial implants, also promotes fibrosis around cardiomyocyte implants. Peri-implant fibrosis, angiogenesis and graft integration will be measured in Tsp2- null and wild type mice. The ability to control graft cell death, graft cell proliferation and peri-implant fibrosis would substantially advance cell-based cardiac repair. These advances also should be applicable to other diseases treatable by cell therapy, such as diabetes, Parkinson disease and spinal cord injury. In addition to potential therapeutic applications, these studies will provide the first systematic analysis of pathways regulating death and proliferation in human cardiomyocytes.